308
Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs Hiroshi Yamasaki Department Johnan-ku,
of Dentistry and Oral Surgery, Fukuoka, 814-01, Japan
School of Medicine,
Fukuoka
University,
7-45-l
Nanakuma,
Hidetaka Sakai Department 872, Japan
of Oral Pathology,
Faculty of Dentistry,
Kyushu University,
3-l-l
Maidashi,
Higashi-ku,
Fukuoka,
Soft tissue reactions to two materials of different microstructure made with porous and dense hydroxyapatite ceramic granules, were compared in this study. Each was implanted subcutaneously in the abdomen of 10 dogs. The implants were removed 1, 3 and 6 months after implantation, and examined by light microscopy. Heterotopic bone formation was only evident around porous hydroxyapatite ceramic granules in 6 of the 10 animals after 3 months, and in all cases after 6 months, but never around dense granules. These findings suggested that cellular differentiation and induction of osteogenesis may be influenced by the physical and chemical factors in the micromilieu provided by porous hydroxyapatite ceramic granules. Keywords: Received
Hydroxyapatite, 12 November
microstructure
1990; revised 24 January
(HA) ceramics implanted in osseous sites bind directly with bone. This material has been applied clinically as a bone substitute’-3. Bone formation around an implanted HA ceramic substitute is generally thought to occur by osteoconduction from surrounding osseous tissues4. However, the extent of bone formation within the structure of porous HA raises the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive material’. In non-osseous sites, on the other hand, no report has been made that HA ceramics promote bone formation. Most investigators believe that this biomaterial is devoid of intrinsic osteoinductive properties’-‘. Recently Heughebaert et aI.” reported that the physico-chemical analyses of porous HA ceramics implanted in hamster soft tissues for 12 months, demonstrated the formation of bone-like material. The purpose of this study was to evaluate the soft tissue response to the subcutaneous placement of porous and dense HA ceramic granules, and specifically to search for any histological evidence of bone formation around them. Hydroxyapatite
Correspondence
Biomateriah
to Dr H. Yamasaki.
1992, Vol. 13 No. 5
osteogenesis,
tissue
1991; accepted
MATERIALS
reaction
23 March 1991
AND METHODS
Materials Two types of HA ceramic granules, porous and dense, were wet synthesized and sintered at 1200’C (Asahi Optical Co., Tokyo, Japan]. These granules ranged in size from 200 to 600 pm and porous granules had a continuous and interconnected microporosity ranging in diameter from 2 to 10 pm (Figure 2). The X-ray diffraction analysis showed that these granules were almost pure hydroxyapatite (>99.9%) without tricalcium phosphate and pirophosphate (Figure 2). The infrared absorption spectroscopy data of these granules showed that the ceramics were not decomposed and there were hydroxyl groups. Methods Ten adult male mongrel dogs anaesthetized with sodium pentobarbital were used in all experiments. The abdomen of each dog was shaved and prepared with a povidoneiodine solution, and six separate subcutaneous pockets were created by blunt dissection. Porous granules were implanted on the left and dense granules were implanted 0 1992 Butterworth-Heinemann Ltd 0142-9612/92/050308-05
Osteogenic
response
H. Yamasaki and H. Sakai
to hydroxyapatites:
309
Figure 3 The hydroxyapatite (HA) mass of the porous-type granules within the subcutis of dog abdomen is shown to be mobile and has a bone-like consistency 3 months after implantation.
RESULTS Macroscopic
observations
Each implanted HA mass of granules was mobile when palpated, and many of the porous type had a bone-like consistency, 3 and 6 months after implantation (Figure 3).
Microscopic Figure 1 Scanning electron photomicrograph of the surface of: a, porous, b, dense hydroxyapatite ceramic granules.
c)
5
s
L
25
26
27
28
29
30
31
L
32
33
34
35
36
37
38
39
40
28(O)
Figure 2 X-ray diffraction pattern of porous hydroxyapatite (HA) ceramic granules, showing that the granules are >99.9% pure hydroxyapatite. That of dense type shows same findings.
on the right side, both in sites far from osseous tissues, The implant quantities were -1 g each. The animals were administered antibiotic prophylaxis orally for 4 d after the implantation procedure. The implants were removed along with the surrounding tissue 1,3 and 6 months after implantation. All specimens were fixed in neutrally buffered formalin, decalcified using the Plank-Rychlo technique, and examined by light microscopy.
observations
Neither porous or dense HA ceramics showed bone formation histologically, 1 month after implantation. These granules were surrounded with fibrous connective tissue and partially multinucleated giant cells appeared in the specimens. Three months after implantation, newly formed bone tissue was revealed around the porous HA ceramics in six of ten animals studied, but no ossification was found at any implantation site of the dense HA ceramic. The HA mass was localized within the subcutis and surrounded with fibrous connective tissue (Figure 4). The newly-formed bone tissue presented features of intramembranous ossification without cartilage formation. The new osseous tissue was primarily immature trabecular bone with intralacunar osteocytes, with obvious osteoblastic rimming [Figure 5). The osteoblasts were seen near the surface of the porous HA ceramics, along with adjacent bone-matrix formation and proliferation of preosteoblast-like cells with large nuclei [Figure 6). The micropores of the HA ceramics were found to be filled with eosinophilic amorphous substance, suggesting a bone matrix. The dense HA ceramics were surrounded by collagen fibres and occasional macrophages and multinucleated giant cells, but in no case was there evidence of bone-matrix formation or calcification at 3 months [Figure 7). Six months after implantation, the specimens from the porous type revealed new bone formation in all animals, and fatty bone marrows were seen among the bone trabeculae in four of ten dogs [Figure 8). However, neither ossification or calcification was found in the specimens from the dense type, and, on the surface of HA ceramics, macrophages and multinucleated giant cells were prominent [Figure 9). Riomaterials
1992, Vol. 13 No. 5
310
Osteogenic response
Figure 4 Specimens, 3 months after implantation, show the porous hydroxyapatite (HA) ceramic granules surrounded with fibrous connective tissue within the subcutis. The arrow in a, indicates the region highlighted in b. New bone formation around the hydroxyapatite (HA) ceramic granules and trabecular structures are demonstrated. P: porous hydroxyapatite (HA) ceramics (original magnification: a, X6; b, X25).
to hvdroxyapatites:
H. Yamasaki and H. Sakai
Figure 6 Differentiating osteoblasts and bone-matrix formation are seen near the porous hydroxyapatite (HA) ceramics. Small arrows in a and b show eosinophilic substance within the micropore of the porous hydroxyapatite (HA) ceramic granules. Large arrow in a shows an preosteoblast-like cell. P: porous hydroxyapatite (HA) ceramics (original magnification: a, x100, b, x50).
D
Figure5 Higher-power views of the new bone around the porous hydroxyapatite (HA) ceramics. Osteoblastic rimming around the new bone and intralacunar osteocytes can be noted (original magnification X100).
Figure 7 Dense hydroxyapatite (HA) ceramics are surrounded by collagen fibres. No osseous tissue is found 3 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).
DISCUSSION Clinically, heterotopic bone formation is encountered as a complication in spinal cord injury and hemiplegia. Experiments have demonstrated heterotopic bone forBiomaterials
1992, Vol. 13 No. 5
mation in animals bearing grafts of urinary tract tissues” and in those with implanted poly(hydroxyethy1 methacrylate) sponge I2. Heterotopic bone formation has also been seen in animals with bone morphogenetic protein
Osteogenic
response
to hydroxyapatites:
311
H. Yamasaki and H. Sakai
Figure8 Specimen from the porous hydroxyapatite (HA) ceramic, 6 months after implantation, showing new bone formation and fatty bone marrow among the bone trabeculae. P: porous hydroxyapatite (HA) ceramics, M: bone marrow (original magnification X25).
Figure9 Macrophages and multinucleated giant cells are prominent on the surface of the de;ise hydroxyapatite (HA) ceramics 6 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).
(BMP) extracted from bone matrix, but the underlying cause and mechanism remain unclear4. Experiments on HA ceramic implantation in soft tissues have been reported, and some have involved longterm observations, although none presented evidence of osteogenesis. Most of these studies dealt with dense HA ceramics’3-‘6, whilst studies of porous HA ceramics comprised short-term observations of less than 3 months5+‘. Heughebaert et aI.” reported that the physicochemical analyses of porous HA ceramic implanted in soft tissues of hamsters for 12 months demonstrated the formation of bone-like material and they thought the HA ceramic promoted new bone formation (osteoinduction). However, they also reported that the new material did not give the histological and morphological characteristics of true bone. Recently we found that the subcutaneous implantation of porous HA ceramic granules in the abdomen of the dog, resulted in heterotopic bone formation, 3 months after implantation17.
However, many reports have appeared concerning experimental implantation of HA ceramics in or on bone’8-24. Some of the reports describe the observation of newly-formed osseous tissue, as well as the differentiation of osteoblasts at the surface of the HA ceramic implant. These reactions have generally been interpreted as a process of extension of bone growth from the surrounding some investigators osseous tissues’8’ 24. However, consider them to be a sign of the osteoinductive processes’” “, and the extent of bone formation within the structure of porous HA has raised the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive materia15. Reddi and Hugginsz5 reported that osteogenic transformation of fibroblasts was profoundly influenced by the geometry of the transformants, such as demineralized bone powder and whole teeth. that physical factors, El Deeb et aL5 suggested including the three-dimensional configuration of porous HA ceramics, were important in bone formation within the material. In this study, osteoblast differentiation and new bone formation at the surface of implanted porous HA ceramic granules were obviously demonstrated in nonosseous sites. The micropores of the HA ceramics were filled with eosinophilic amorphous substance, suggesting the ingrowth of connective tissue9 or unknown organic materials’“. On the contrary, there was no evidence of newly-formed osseous tissue at the implantation site of dense HA ceramic granules. These findings suggest that physical and chemical factors in the micromilieu provided by implanted HA ceramic granules possessing micropores exert an effect on osteogenesis. We speculate that they may be due to calcium phosphates leaching out of the porous granules or the ion-exchange reactions in the adjacent tissue. The release of co-phosphate can depend not only on the porosity of HA samples, but also on other accompanying amorphous material not detectable from X-ray analyses or infrared spectroscopy.
ACKNOWLEDGEMENT The authors thank Professor H. Miyako for supporting this study. M. Kishimoto for technical assistance, and Y. Hirayama (Asahi Optical Co., Tokyo, Japan) for preparing the implant materials used in this study.
REFERENCES Denissen, H.W. and De Groot, K., Immediate dental root implants from synthetic dense calcium hydroxyapatite, 1. Prosthet. Dent. 1979, 42, 551-556 Block, MS. and Kent, J.N., Long-term radiographic evaluation of hydroxyapatite augmentation of deficient mandibular alveolar ridges, 1. Oral Maxiflofac. Surg. 1984, 42, 793-796 Donath, K., Rohrer, M.D. and Beck-Mannagetta, J,, A histologic evaluation of a mandibular cross section one year after augmentation with hydroxyapatite particles, Oral Surg. Oral Med. Oral Pathol. 1987, 63, 651-655 Biomaterials
1992,
Vol.
13 No. 5
312
10
11
12
13
14
Osteogenic Urist, M.R., Heterotopic bone formation, inFundamental and ClinicalBone Physiology (Ed. M.R. Urist), Lippincott, Philadelphia, USA, 1960, pp 369-393 El Deeb, M.E., Hosny, M. and Sharawy, M., Osteogenesis in composite grafts of allogenic demineralized bone powder and porous hydroxylapatite, J. Oral Maxillofac. Surg. 1969, 47,50-56 Piecuch, J.F., Extraskeletal implantation of a porous hydroxyapatite ceramic, J. Dent. Res. 1982,61, 145a1460 Shibata, S., An experimental study for effect of implant of fresh autogenous bone marrow mixed with hydroxylapatite particles in subcutaneous tissue and jaw bone defects, Jpn. J. Oral Maxillofac. Surg. 19aa,34,217a-21a7 (in Japanese) Negami, S., Histological observations on muscle tissue reaction to porous hydroxyapatite sintered body in rats, J. Jpn. Ortbop. Assoc. 1966, 62, 65-94 (in Japanese) Klein, C.A.P.T., Lubbe, H.B.M., De Groot, K. and Hooff, A., Biodegradation behaviour of various calcium phosphate materials in subcutaneous tissue, in Materials Science Monographs 17: Ceramics in Surgery (Ed. P. Vincenzini), Elsevier, Amsterdam, The Netherlands, 1983, pp 105-115 Heughebaert, M., Le Geros, R.Z., Gineste, M., Guilhem, A. and Bonel, G., Physicochemical characterization of deposits associated with HA ceramics implanted in nonosseous sites, J. Biomed. Mater. Res. 1966, 22, 257-268 Huggins, C.B., The formation of bone under the influence of epithelium of the urinary tract, Arch. Surg. 1931, 22, 377-408 Winter, G.D. and Simpson, B.J., Heterotopic bone formed in a synthetic sponge in the skin of young pigs, Nature 1969, 223, 66-90 Denissen, H.W. and De Groot, K., Tissue response to dense apatite implants in rats, J. Biomed. Mater. Res. 1960,14,713-721 Drobeck, H.P., Rothstein, S.S., Gumaer, K.I., Sherer, A.D. and Slighter, R.G., Histologic observation of soft tissue responses to implanted, multifaceted particles and
Biomaterials
1992, Vol. 13 No. 5
response
15
16
17
la 19
20
21
22
23
24
25
26
to hydroxyapatites:
H. Yamasaki and H. Sakai
discs of hydroxylapatite, J. Oral Maxillofac. Surg. 1984, 42,143-149 Misiek, D.J., Kent, J.N. and Carr, R.F., Soft tissue responses to hydroxylapatite particles of different shapes, J. Oral Maxillofac. Surg. 1964, 42, 150-160 Harvey,W.K., Pincock, J.L., Matukas, V.J. and Lemons, J.E., Evaluation of a subcutaneously implanted hydroxylapatite-Avitene mixture in rabbits, J. Oral Maxillofac. Surg. i985,43,277-280 Yamasaki, H., Heterotopic bone formation around porous hydroxyapatite ceramics in the subcutis of dogs, Jpn. J. Oral Viol. 1990, 32, 190-192 Jarcho, M., Calcium phosphate ceramics as hard tissue prosthetics, Clin. Orthop. 1981,157,259-278 Ogiso, M., Formation and calcification of bone tissue on the surface of apatite ceramics, J. Stomatol. Sot. Jpn. 1963, 50, l-22 (in Japanese] Piecuch, J.F., Topazian, R.G., Skoly, S. and Wolf, S., Experimental ridge augmentation with porous hydroxyapatite implants, J. Dent. Res. 1963, 62,148-154 Katthagen, B.D. and Mittelmeier, H., Experimental animal investigation of bone regeneration with collagenapatite, Arch. Ortbop. Trauma. Surg. 1984,103,291-302 Kenny, E.B., Lekovic, V., Sa Ferreira, J.C., Han, T., Dimitrijevic, B. and Carranza, Jr. F.A., Bone formation within porous hydroxylapatite implants in human periodontal defects, J. Periodontal. 1966, 57, 76-63 Gumaer, K.I., Sherer, A.D., Slighter, R.G., Rothstein, S.S. and Drobeck, H.P., Tissue response in dogs to dense hydroxylapatite implantation in the femur, J, Oral Maxillofac. Surg. 1966, 44, 616-627 Bucholz, R.W., Carton, A. and Holmes, R.H., Hydroxyapatite and tricalcium phosphate bone graft substitutes, Orthop. Clin. North Am. 1967, 18,323-334 Reddi, A.H. and Huggins, C.B., Influence of geometry of transplanted tooth and bone on transformation of fibroblasts, Proc. Sot. Exp. Biol. Med. 1973,143,634-637 Krajewski, A., Ravaglioli, A., Mongiorgi, R. and Moroni, A., Mineralization and calcium fixation within a porous apatite ceramic material after implantation in the femur of rabbits, J. Biomed. Mater. Res. 1966, 22, 445-457
Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs Hiroshi Yamasaki Department Johnan-ku,
of Dentistry and Oral Surgery, Fukuoka, 814-01, Japan
School of Medicine,
Fukuoka
University,
7-45-l
Nanakuma,
Hidetaka Sakai Department 872, Japan
of Oral Pathology,
Faculty of Dentistry,
Kyushu University,
3-l-l
Maidashi,
Higashi-ku,
Fukuoka,
Soft tissue reactions to two materials of different microstructure made with porous and dense hydroxyapatite ceramic granules, were compared in this study. Each was implanted subcutaneously in the abdomen of 10 dogs. The implants were removed 1, 3 and 6 months after implantation, and examined by light microscopy. Heterotopic bone formation was only evident around porous hydroxyapatite ceramic granules in 6 of the 10 animals after 3 months, and in all cases after 6 months, but never around dense granules. These findings suggested that cellular differentiation and induction of osteogenesis may be influenced by the physical and chemical factors in the micromilieu provided by porous hydroxyapatite ceramic granules. Keywords: Received
Hydroxyapatite, 12 November
microstructure
1990; revised 24 January
(HA) ceramics implanted in osseous sites bind directly with bone. This material has been applied clinically as a bone substitute’-3. Bone formation around an implanted HA ceramic substitute is generally thought to occur by osteoconduction from surrounding osseous tissues4. However, the extent of bone formation within the structure of porous HA raises the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive material’. In non-osseous sites, on the other hand, no report has been made that HA ceramics promote bone formation. Most investigators believe that this biomaterial is devoid of intrinsic osteoinductive properties’-‘. Recently Heughebaert et aI.” reported that the physico-chemical analyses of porous HA ceramics implanted in hamster soft tissues for 12 months, demonstrated the formation of bone-like material. The purpose of this study was to evaluate the soft tissue response to the subcutaneous placement of porous and dense HA ceramic granules, and specifically to search for any histological evidence of bone formation around them. Hydroxyapatite
Correspondence
Biomateriah
to Dr H. Yamasaki.
1992, Vol. 13 No. 5
osteogenesis,
tissue
1991; accepted
MATERIALS
reaction
23 March 1991
AND METHODS
Materials Two types of HA ceramic granules, porous and dense, were wet synthesized and sintered at 1200’C (Asahi Optical Co., Tokyo, Japan]. These granules ranged in size from 200 to 600 pm and porous granules had a continuous and interconnected microporosity ranging in diameter from 2 to 10 pm (Figure 2). The X-ray diffraction analysis showed that these granules were almost pure hydroxyapatite (>99.9%) without tricalcium phosphate and pirophosphate (Figure 2). The infrared absorption spectroscopy data of these granules showed that the ceramics were not decomposed and there were hydroxyl groups. Methods Ten adult male mongrel dogs anaesthetized with sodium pentobarbital were used in all experiments. The abdomen of each dog was shaved and prepared with a povidoneiodine solution, and six separate subcutaneous pockets were created by blunt dissection. Porous granules were implanted on the left and dense granules were implanted 0 1992 Butterworth-Heinemann Ltd 0142-9612/92/050308-05
Osteogenic
response
H. Yamasaki and H. Sakai
to hydroxyapatites:
309
Figure 3 The hydroxyapatite (HA) mass of the porous-type granules within the subcutis of dog abdomen is shown to be mobile and has a bone-like consistency 3 months after implantation.
RESULTS Macroscopic
observations
Each implanted HA mass of granules was mobile when palpated, and many of the porous type had a bone-like consistency, 3 and 6 months after implantation (Figure 3).
Microscopic Figure 1 Scanning electron photomicrograph of the surface of: a, porous, b, dense hydroxyapatite ceramic granules.
c)
5
s
L
25
26
27
28
29
30
31
L
32
33
34
35
36
37
38
39
40
28(O)
Figure 2 X-ray diffraction pattern of porous hydroxyapatite (HA) ceramic granules, showing that the granules are >99.9% pure hydroxyapatite. That of dense type shows same findings.
on the right side, both in sites far from osseous tissues, The implant quantities were -1 g each. The animals were administered antibiotic prophylaxis orally for 4 d after the implantation procedure. The implants were removed along with the surrounding tissue 1,3 and 6 months after implantation. All specimens were fixed in neutrally buffered formalin, decalcified using the Plank-Rychlo technique, and examined by light microscopy.
observations
Neither porous or dense HA ceramics showed bone formation histologically, 1 month after implantation. These granules were surrounded with fibrous connective tissue and partially multinucleated giant cells appeared in the specimens. Three months after implantation, newly formed bone tissue was revealed around the porous HA ceramics in six of ten animals studied, but no ossification was found at any implantation site of the dense HA ceramic. The HA mass was localized within the subcutis and surrounded with fibrous connective tissue (Figure 4). The newly-formed bone tissue presented features of intramembranous ossification without cartilage formation. The new osseous tissue was primarily immature trabecular bone with intralacunar osteocytes, with obvious osteoblastic rimming [Figure 5). The osteoblasts were seen near the surface of the porous HA ceramics, along with adjacent bone-matrix formation and proliferation of preosteoblast-like cells with large nuclei [Figure 6). The micropores of the HA ceramics were found to be filled with eosinophilic amorphous substance, suggesting a bone matrix. The dense HA ceramics were surrounded by collagen fibres and occasional macrophages and multinucleated giant cells, but in no case was there evidence of bone-matrix formation or calcification at 3 months [Figure 7). Six months after implantation, the specimens from the porous type revealed new bone formation in all animals, and fatty bone marrows were seen among the bone trabeculae in four of ten dogs [Figure 8). However, neither ossification or calcification was found in the specimens from the dense type, and, on the surface of HA ceramics, macrophages and multinucleated giant cells were prominent [Figure 9). Riomaterials
1992, Vol. 13 No. 5
310
Osteogenic response
Figure 4 Specimens, 3 months after implantation, show the porous hydroxyapatite (HA) ceramic granules surrounded with fibrous connective tissue within the subcutis. The arrow in a, indicates the region highlighted in b. New bone formation around the hydroxyapatite (HA) ceramic granules and trabecular structures are demonstrated. P: porous hydroxyapatite (HA) ceramics (original magnification: a, X6; b, X25).
to hvdroxyapatites:
H. Yamasaki and H. Sakai
Figure 6 Differentiating osteoblasts and bone-matrix formation are seen near the porous hydroxyapatite (HA) ceramics. Small arrows in a and b show eosinophilic substance within the micropore of the porous hydroxyapatite (HA) ceramic granules. Large arrow in a shows an preosteoblast-like cell. P: porous hydroxyapatite (HA) ceramics (original magnification: a, x100, b, x50).
D
Figure5 Higher-power views of the new bone around the porous hydroxyapatite (HA) ceramics. Osteoblastic rimming around the new bone and intralacunar osteocytes can be noted (original magnification X100).
Figure 7 Dense hydroxyapatite (HA) ceramics are surrounded by collagen fibres. No osseous tissue is found 3 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).
DISCUSSION Clinically, heterotopic bone formation is encountered as a complication in spinal cord injury and hemiplegia. Experiments have demonstrated heterotopic bone forBiomaterials
1992, Vol. 13 No. 5
mation in animals bearing grafts of urinary tract tissues” and in those with implanted poly(hydroxyethy1 methacrylate) sponge I2. Heterotopic bone formation has also been seen in animals with bone morphogenetic protein
Osteogenic
response
to hydroxyapatites:
311
H. Yamasaki and H. Sakai
Figure8 Specimen from the porous hydroxyapatite (HA) ceramic, 6 months after implantation, showing new bone formation and fatty bone marrow among the bone trabeculae. P: porous hydroxyapatite (HA) ceramics, M: bone marrow (original magnification X25).
Figure9 Macrophages and multinucleated giant cells are prominent on the surface of the de;ise hydroxyapatite (HA) ceramics 6 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).
(BMP) extracted from bone matrix, but the underlying cause and mechanism remain unclear4. Experiments on HA ceramic implantation in soft tissues have been reported, and some have involved longterm observations, although none presented evidence of osteogenesis. Most of these studies dealt with dense HA ceramics’3-‘6, whilst studies of porous HA ceramics comprised short-term observations of less than 3 months5+‘. Heughebaert et aI.” reported that the physicochemical analyses of porous HA ceramic implanted in soft tissues of hamsters for 12 months demonstrated the formation of bone-like material and they thought the HA ceramic promoted new bone formation (osteoinduction). However, they also reported that the new material did not give the histological and morphological characteristics of true bone. Recently we found that the subcutaneous implantation of porous HA ceramic granules in the abdomen of the dog, resulted in heterotopic bone formation, 3 months after implantation17.
However, many reports have appeared concerning experimental implantation of HA ceramics in or on bone’8-24. Some of the reports describe the observation of newly-formed osseous tissue, as well as the differentiation of osteoblasts at the surface of the HA ceramic implant. These reactions have generally been interpreted as a process of extension of bone growth from the surrounding some investigators osseous tissues’8’ 24. However, consider them to be a sign of the osteoinductive processes’” “, and the extent of bone formation within the structure of porous HA has raised the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive materia15. Reddi and Hugginsz5 reported that osteogenic transformation of fibroblasts was profoundly influenced by the geometry of the transformants, such as demineralized bone powder and whole teeth. that physical factors, El Deeb et aL5 suggested including the three-dimensional configuration of porous HA ceramics, were important in bone formation within the material. In this study, osteoblast differentiation and new bone formation at the surface of implanted porous HA ceramic granules were obviously demonstrated in nonosseous sites. The micropores of the HA ceramics were filled with eosinophilic amorphous substance, suggesting the ingrowth of connective tissue9 or unknown organic materials’“. On the contrary, there was no evidence of newly-formed osseous tissue at the implantation site of dense HA ceramic granules. These findings suggest that physical and chemical factors in the micromilieu provided by implanted HA ceramic granules possessing micropores exert an effect on osteogenesis. We speculate that they may be due to calcium phosphates leaching out of the porous granules or the ion-exchange reactions in the adjacent tissue. The release of co-phosphate can depend not only on the porosity of HA samples, but also on other accompanying amorphous material not detectable from X-ray analyses or infrared spectroscopy.
ACKNOWLEDGEMENT The authors thank Professor H. Miyako for supporting this study. M. Kishimoto for technical assistance, and Y. Hirayama (Asahi Optical Co., Tokyo, Japan) for preparing the implant materials used in this study.
REFERENCES Denissen, H.W. and De Groot, K., Immediate dental root implants from synthetic dense calcium hydroxyapatite, 1. Prosthet. Dent. 1979, 42, 551-556 Block, MS. and Kent, J.N., Long-term radiographic evaluation of hydroxyapatite augmentation of deficient mandibular alveolar ridges, 1. Oral Maxiflofac. Surg. 1984, 42, 793-796 Donath, K., Rohrer, M.D. and Beck-Mannagetta, J,, A histologic evaluation of a mandibular cross section one year after augmentation with hydroxyapatite particles, Oral Surg. Oral Med. Oral Pathol. 1987, 63, 651-655 Biomaterials
1992,
Vol.
13 No. 5
312
10
11
12
13
14
Osteogenic Urist, M.R., Heterotopic bone formation, inFundamental and ClinicalBone Physiology (Ed. M.R. Urist), Lippincott, Philadelphia, USA, 1960, pp 369-393 El Deeb, M.E., Hosny, M. and Sharawy, M., Osteogenesis in composite grafts of allogenic demineralized bone powder and porous hydroxylapatite, J. Oral Maxillofac. Surg. 1969, 47,50-56 Piecuch, J.F., Extraskeletal implantation of a porous hydroxyapatite ceramic, J. Dent. Res. 1982,61, 145a1460 Shibata, S., An experimental study for effect of implant of fresh autogenous bone marrow mixed with hydroxylapatite particles in subcutaneous tissue and jaw bone defects, Jpn. J. Oral Maxillofac. Surg. 19aa,34,217a-21a7 (in Japanese) Negami, S., Histological observations on muscle tissue reaction to porous hydroxyapatite sintered body in rats, J. Jpn. Ortbop. Assoc. 1966, 62, 65-94 (in Japanese) Klein, C.A.P.T., Lubbe, H.B.M., De Groot, K. and Hooff, A., Biodegradation behaviour of various calcium phosphate materials in subcutaneous tissue, in Materials Science Monographs 17: Ceramics in Surgery (Ed. P. Vincenzini), Elsevier, Amsterdam, The Netherlands, 1983, pp 105-115 Heughebaert, M., Le Geros, R.Z., Gineste, M., Guilhem, A. and Bonel, G., Physicochemical characterization of deposits associated with HA ceramics implanted in nonosseous sites, J. Biomed. Mater. Res. 1966, 22, 257-268 Huggins, C.B., The formation of bone under the influence of epithelium of the urinary tract, Arch. Surg. 1931, 22, 377-408 Winter, G.D. and Simpson, B.J., Heterotopic bone formed in a synthetic sponge in the skin of young pigs, Nature 1969, 223, 66-90 Denissen, H.W. and De Groot, K., Tissue response to dense apatite implants in rats, J. Biomed. Mater. Res. 1960,14,713-721 Drobeck, H.P., Rothstein, S.S., Gumaer, K.I., Sherer, A.D. and Slighter, R.G., Histologic observation of soft tissue responses to implanted, multifaceted particles and
Biomaterials
1992, Vol. 13 No. 5
response
15
16
17
la 19
20
21
22
23
24
25
26
to hydroxyapatites:
H. Yamasaki and H. Sakai
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